The invention relates to therapeutic compounds and methods for inhibiting angiogenesis.
Angiogenesis
Angiogenesis is the process in which new blood vessels grow into an area which lacks a sufficient blood supply. Angiogenesis commences with the erosion of the basement membrane surrounding endothelial cells and pericytes forming capillary blood vessels. Erosion of the basement membrane is triggered by enzymes released by endothelial cells and leukocytes. The endothelial cells then migrate through the eroded basement membrane when induced by angiogenic stimulants. The migrating cells form a xe2x80x9csproutxe2x80x9d off the parent blood vessel. The migrating endothelial cells proliferate, and the sprouts merge to form capillary loops, thus forming a new blood vessel.
Angiogenesis can occur under certain normal conditions in mammals such as in wound healing, in fetal and embryonic development, and in the formation of the corpus luteum, endometrium and placenta. Angiogenesis also occurs in certain disease states such as in tumor formation and expansion, or in the retina of patients with certain ocular disorders. Angiogenesis can also occur in a rheumatoid joint, hastening joint destruction by allowing an influx of leukocytes with subsequent release of inflammatory mediators.
The evidence for the role of angiogenesis in tumor growth was extensively reviewed by O""Reilly and Folkman in U.S. Pat. No. 5,639,725, the entire disclosure of which is incorporated herein by reference. It is now generally accepted that the growth of tumors is critically dependent upon this process. Primary or metastatic tumor foci are unable to achieve a size of more than approximately 2 mm in the absence of neovascularization. Serial evaluation of transgenic mice predisposed to develop neoplasms has demonstrated that neovascularization of premalignant lesions precedes their evolution into tumors (Folkman et al., Nature 339:58-61, 1989), and that inhibition of angiogenesis delays the growth of such lesions, as well as their assumption of a malignant phenotype (Hanahan et al., Cell 86:353-364, 1996). In humans, several studies have demonstrated that increased density of microvessels within a tumor is associated with a poor clinical outcome (Weidner et al., J Natl Cancer Inst 84:1875-1887, 1992).
An emerging paradigm is that proteolytic fragments of plasma or extracellular matrix proteins regulate angiogenesis. To date, several polypeptides with such activities have been identified. These include angiostatin, which contains kringles 1-4 plasminogen (O""Reilly et al., Cell 79:315-328, 1994), endostatin, a 20 kD C-terminal fragment of collagen XVIII (O""Reilly et al., Cell 88:277-285, 1997), PEX, the hemopexin domain of matrix metalloprotease 2 (Brooks et al., Cell 92:391-400, 1998), the C-terminal 16 kD fragment of prolactin (Clapp et al., Endocrinol 133:1292-1299, 1993) and a 29 kD fragment of fibronectin (Homandberg et al., Am J Pathol 120:327-332; 1985). In addition, both intact thrombospondin 1 as well as peptides derived from its procollagen domain and properdin-like type-1 repeats express potent anti-angiogenic activity (Good et al., Proc Nat Acad Sci USA 87:6624-6628,1990); Tolsma et al., J Cell Biol 122:497-511, 1993. In preclinical models, several of these fragments inhibited tumor growth, and some induced tumor regression and dormancy (Boehm et al., Nature 390:404-407, 1997).
High Molecular Weight Kininogen
High molecular weight kininogen (HK) is a 120 kD glycoprotein containing heavy and light chains, comprised of domains 1 through 3, and 5 and 6, respectively (Kaplan et al., Blood 70:1-15, 1987). The heavy and light chains are linked by domain 4, which contains bradykinin, a nonapeptide which mediates several events including NO-dependent vasodilation (Weimer et al., J Pharm Exp Therapeutics 262:729-733, 1992). HK (also referred to as xe2x80x9csingle chain high molecular weight kininogenxe2x80x9d) binds with high affinity to endothelial cells, where it is cleaved to two-chain high molecular weight kininogen (HKa) by plasma kallikrein. Bradykinin is released from HK through cleavage mediated by plasma kallikrein (Kaplan et al., Blood 70:1-15, 1987). This event occurs on the surface of endothelial cells following the activation of prekallikrein to kallikrein by an endothelial cell protease (Motta et al., Blood 91:515-528, 1998). Cleavage of HK to form HKa and release bradykinin occurs between Lys(362) and Arg(363). HKa contains a 62 kD heavy chain and a 56 kD light chain linked by a disulfide bond.
Conversion of HK to HKa is accompanied by a dramatic structural rearrangement, which has been demonstrated using rotary shadowing electron microscopy (Weisel et al. J. Biol Chem 269:10100-10106, 1994). HKa, but not HK, has been shown to inhibit the adhesion of endothelial and other cell types to vitronectin (Asakura, J. Cell Biol 116:465-476, 1992).
Although the binding of HK to endothelial cells has been well characterized, comparatively little attention has been devoted to endothelial cell binding of Ha. Furthermore, although binding of bradykinin to endothelial cells induces well-defined responses, functional consequences of the direct binding of HKa have not been reported.
The compounds of the present invention are in the form of peptides which possess anti-angiogenic activity.
In all embodiments, the peptide may optionally comprise an amino-terminal and/or carboxy-terminal protecting group.
A compound of the formula X1-His-Lys-X-Lys-X2 (hereinafter xe2x80x9cX1-His-Lys-X-Lys-X2 peptidexe2x80x9d) is provided wherein
X is any amino acid,
X1 is from zero to twelve amino acids, more preferably from zero to six amino acids, most preferably from zero to three amino acids, and
X2 is from zero to twelve amino acids, more preferably from zero to six amino acids, most preferably from zero to three amino acids.
Preferably, X is an amino acid having a nonpolar side chain, i.e., Ala, Leu, Ile, Val, Pro, Phe, Trp, or Met; or X is an amino acid having a polar side group which is uncharged at pH 6.0 to 7.0, the zone of physiological pH, i.e., Ser, Thr, Tyr, Asn, Gln, Cys, or Gly. Most preferably, X is Asn, Phe or His.
Preferred compounds comprise fragments of HK. In one group of such preferred compounds,
X1 is
(i) zero amino acids, or
(ii) the segment His-Gly-His-Glu-Gln-Gln-His-Gly-Leu-Gly-His-Gly (SEQ ID NO:1) or N-terminal truncation fragment thereof containing at least one amino acid, and
X2 is
(i) zero amino acids, or
(ii) the segment Leu-Asp-Asp-Asp-Leu-Glu-His-Gln-Gly-Gly-His-Val (SEQ ID NO:2), or C-terminal truncation fragment thereof containing at least one amino acid.
In another group of such preferred compounds,
X1 is
(i) zero amino acids, or
(ii) the segment Gly-His-Lys-His-Lys-His-Gly-His-Gly-His-Gly-Lys (SEQ ID NO:3) or N-terminal truncation fragment thereof containing at least one amino acid, and
X2 is
(i) zero amino acids, or
(ii) the segment Gly-Lys-Lys-Asn-Gly-Lys-His-Asn-Gly-Trp-Lys-Thr (SEQ ID NO:4) or C-terminal truncation fragment thereof containing at least one amino acid.
According to a further preferred embodiment of the invention, the compound has a substantial amino acid homology to either the amino acid sequence His-Gly-His-Glu-Gln-Gln-His-Gly-Leu-Gly-His-Gly-His-Lys-Phe-Lys-Leu-Asp-Asp-Asp-Leu-Glu-His-Gln-Gly-Gly-His-Val (SEQ ID NO:5), or the amino acid sequence Gly-His-Lys-His-Lys-His-Gly-His-Gly-His-Gly-Lys-His-Lys-Asn-Lys-Gly-Lys-Lys-Asn-Gly-Lys-His-Asn-Gly-Trp-Lys-Thr (SEQ ID NO:6).
Exemplary and preferred compounds include:
(a) His-Gly-His-Glu-Gln-Gln-His-Gly-Leu-Gly-His-Gly-His-Lys-Phe-Lys-Leu-Asp-Asp-Asp-Leu-Glu-His-Gln-Gly-Gly-His-Val (SEQ ID NO:5);
(b) Gly-His-Lys-Phe-Lys-Leu-Asp-Asp-Asp-Leu-Glu-His-Gln-Gly-Gly-His (SEQ ID NO:7);
(c) Gly-His-Lys-His-Lys-His-Gly-His-Gly-His-Gly-Lys-His-Lys-Asn-Lys-Gly-Lys-Lys-Asn-Gly-Lys-His-Asn-Gly-Trp-Lys-Thr (SEQ ID NO:6);
(d) Lys-His-Gly-His-Gly-His-Gly-Lys-His-Lys-Asn-Lys-Gly-Lys-Lys-Asn (SEQ ID NO:8); and
(e) His-Lys-Asn-Lys-Gly-Lys-Lys-Asn-Gly-Lys-His-Asn-Gly-Trp-Lys-Thr (SEQ ID NO:9).
The invention also encompasses a method of inhibiting endothelial cell proliferation comprising contacting endothelial cells with HK, HKa or a X1-His-Lys-X-Lys-X2 peptide.
The invention also encompasses a method of inducing apoptosis of endothelial cells comprising contacting endothelial cells with HK, HKa or a X1-His-Lys-X-Lys-X2 peptide.
The invention is also a composition comprising a pharmaceutically effective carrier and HK, HKa or a X1-His-Lys-X-Lys-X2 peptide.
The invention is also a method of inhibiting angiogenesis in a mammal in need of such treatment comprising administering to said mammal a therapeutically effective amount of a composition comprising a pharmaceutically effective carrier and HK, HKa or a X1-His-Lys-X-Lys-X2 peptide. The mammal treated is preferably a human being.
Other aspects and advantages of the present invention are described in the drawings and in the following detailed description of the preferred embodiments thereof.
Abbreviations and Short Forms
The following abbreviations and short forms are used in this specification.
xe2x80x9cBFGFxe2x80x9d is recombinant human basic fibroblast growth factor.
xe2x80x9cHKxe2x80x9d means the mature form of high molecular weight kininogen, and any allelic variations thereof. By xe2x80x9cmaturexe2x80x9d is meant the post-translationally-modified form of HK which results from cleavage of an eighteen amino acid leader from the initially translated molecule. All numbering with respect to amino acid positions of HK is from the N-terminus of the mature form as position 1. xe2x80x9cHKxe2x80x9d is synonymous with xe2x80x9csingle chain HKxe2x80x9d, the mature form of high molecular weight kininogen prior to cleavage by kallikrein and the formation of two chain high molecular weight kininogen.
xe2x80x9cHKaxe2x80x9d means two-chain high molecular weight kininogen, the product of kallikrein cleavage of mature high molecular weight kininogen, and any allelic variations thereof.
xe2x80x9cHDMVECxe2x80x9d means human dermal microvascular endothelial cells.
xe2x80x9cHGFxe2x80x9d means hepatocyte growth factor.
xe2x80x9cHUVECxe2x80x9d means human umbilical vein endothelial cell
xe2x80x9cPDGFxe2x80x9d is platelet-derived growth factor.
xe2x80x9cTGF-xcex2xe2x80x9d is transforming growth factor-xcex2.
xe2x80x9cVEGFxe2x80x9d means vascular endothelial cell growth factor.
xe2x80x9cX1-His-Lys-X-Lys-X2 peptidexe2x80x9d means a compound of the indicated formula wherein X, X1 and X2 are defined as above.
Amino Acid Abbreviations
The nomenclature used to describe polypeptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the lest and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by a one-letter or three-letter designation, corresponding to the trivial name of the amino acid, in accordance with the following schedule:
Definitions
The following definitions, of terms used throughout the specification, are intended as an aid to understanding the scope and practice of the present invention.
xe2x80x9cAngiogenesisxe2x80x9d means the generation of new blood vessels into a tissue or organ.
xe2x80x9cApoptosisxe2x80x9d means a process of programmed cell death.
A xe2x80x9cpeptidexe2x80x9d is a compound comprised of amino acid residues covalently linked by peptide bonds.
The expression xe2x80x9camino acidxe2x80x9d as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. xe2x80x9cNatural amino acidxe2x80x9d means any of the twenty primary, naturally occurring amino acids which typically form peptides, polypeptides, and proteins. xe2x80x9cSynthetic amino acidxe2x80x9d means any other amino acid, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, xe2x80x9csynthetic amino acidxe2x80x9d also encompasses chemically modified amino acids, including but not limited to salts, derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide""s circulating half life without adversely affecting their activity. Additionally, a disulfide linkage may be present or absent in the peptides of the invention, as long as anti-angiogenic activity is maintained.
Amino acids have the following general structure: 
Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group. Peptides comprising a large number of amino acids are sometimes called xe2x80x9cpolypeptidesxe2x80x9d. The amino acids of the peptides described herein and in the appended claims are understood to be either D or L amino acids with L amino acids being preferred.
xe2x80x9cHomologyxe2x80x9d means similarity of sequence reflecting a common evolutionary origin. Peptides or proteins are said to have homology, or similarity, if a substantial number of their amino acids are either (1) identical, or (2) have a chemically similar R side chain. Nucleic acids are said to have homology if a substantial number of their nucleotides are identical.
As used herein, xe2x80x9cprotectedxe2x80x9d with respect to a terminal amino group refers to a terminal amino group of a peptide, which terminal amino group is coupled with any of various amino-terminal protecting groups traditionally employed in peptide synthesis. Such protecting groups include, for example, acyl protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxycarbonyl; and aliphatic urethane protecting groups, for example, tert-butoxycarbonyl or adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides, vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitable protecting groups.
As used herein, xe2x80x9cprotectedxe2x80x9d with respect to a terminal carboxyl group refers to a terminal carboxyl group of a peptide, which terminal carboxyl group is coupled with any of various carboxyl-terminal protecting groups. Such protecting groups include, for example, tert-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond.
xe2x80x9cSubstantial amino acid sequence homologyxe2x80x9d means an amino acid sequence homology greater than about 30%, preferably greater than about60%, more preferably greater than about 80%, and most preferably greater than about 90%.
By xe2x80x9cN-terminal truncation fragmentxe2x80x9d with respect to an amino acid sequence is meant a fragment obtained from a parent sequence by removing one or more amino acids from the N-terminus thereof.
By xe2x80x9cC-terminal truncation fragmentxe2x80x9d with respect to an amino acid sequence is meant a fragment obtained from a parent sequence by removing one or more amino acids from the C-terminus thereof.